Both thapsigargin- and tunicamycin-induced endoplasmic reticulum stress increases expression of Hrd1 in IRE1-dependent fashion.

OBJECTIVES: We have investigated the impact of endoplasmic reticulum (ER) stress, which is often implicated in neurodegenerative diseases, on the expression of Hrd1, an E3 ubiquitin ligase that plays a central role in the process of ER-associated degradation (ERAD). METHODS: SH-SY5Y neuroblastoma cells, a frequently used model for studying neurotoxicity in dopaminergic neurons and the mechanisms of neurodegeneration associated with Parkinson's disease, and parental SK-N-SH cells were studied. RESULTS: We demonstrate that ER stress, induced by thapsigargin or tunicamycin, correlates with the increased expression of Hrd1 in both SH-SY5Y and SK-N-SH cells. Inhibition of PERK does not significantly suppress the thapsigargin- or tunicamycin-induced expression of Hrd1. Nevertheless, PERK inhibition has a positive effect on the survival of SH-SY5Y cells treated with thapsigargin but not on those treated with tunicamycin. Inhibition of IRE1 associated with the inhibition of XBP1 splicing does not affect the survival of SH-SY5Y cells treated with either thapsigargin or tunicamycin but results in the complete suppression of both the thapsigargin- and tunicamycin-induced expression of Hrd1. DISCUSSION: Thus, the ER-stress-induced expression of Hrd1 in SH-SY5Y depends on Hrd1 transcription activation, which is a consequence of IRE1 but not of PERK activation.

Cobalt chloride affects the death of SH-SY5Y cells induced by inhibition of ubiquitin proteasome system. Role of heat shock protein 70 and caspase 3.

The aim our study was to investigate protective effect of cobalt chloride (CoCl2) in the model of proteasome stress of neuroblastoma SH-SY5Y cells induced by bortezomib, an inhibitor of 26S proteasome. We have focused our interests on Hsp70 and activation of caspase 3. Finally, we have compared the effect of CoCl2 with an effect of the pre-treatment of the cells with 17-AAG, an inhibitor of Hsp90 that is capable to induce expression of Hsp70, or with IOX2, an inhibitor of isoform 2 of prolyl hydroxylase that increases stability of hypoxia inducible factor 1α (HIF1α). Pre-treatment of SH-SY5Y cells for 24 h with CoCl2, at concentrations of 150 or 250 µmol/l, and with 17-AAG at concentration 1 µmol/l but not with IOX2 at concentration 100 µmol/l, was associated with significantly increased expression of Hsp70. We have shown that pre-treatment of SH-SY5Y cells with CoCl2 but not with 17-AAG or IOX2 was associated with significant delay of the cell death induced by proteasome stress. CoCl2-mediated effect was consistent with inhibition of bortezomib-induced caspase 3 activation in the cells pre-treated with CoCl2. Despite established neuroprotective properties of Hsp70 our results do not provide strong evidence that the effect of CoCl2 could be mainly attributed to the ability of CoCl2 to induce expression of Hsp70 and other mechanisms have to be considered.

Proteasome Stress Triggers Death of SH-SY5Y and T98G Cells via Different Cellular Mechanisms.

Overload or dysfunction of ubiquitin-proteasome system (UPS) is implicated in mechanisms of neurodegeneration associated with neurodegenerative diseases, e.g. Parkinson and Alzheimer disease, and ischemia-reperfusion injury. The aim of this study was to investigate the possible association between viability of neuroblastoma SH-SY5Y and glioblastoma T98G cells treated with bortezomib, inhibitor of 26S proteasome, and accumulation of ubiquitin-conjugated proteins with respect to direct cytotoxicity of aggregates of ubiquitin-conjugated proteins. Bortezomib-induced death of SH-SY5Y cells was documented after 24 h of treatment while death of T98G cells was delayed up to 48 h. Already after 4 h of treatment of both SH-SY5Y and T98G cells with bortezomib, increased levels of both ubiquitin-conjugated proteins with molecular mass more than 150 kDa and Hsp70 were observed whereas Hsp90 was elevated in T98G cells and decreased in SH-SY5Y cells. With respect to the cell death mechanism, we have documented bortezomib-induced activation of caspase 3 in SH-SY5Y cells that was probably a result of increased expression of pro-apoptotic proteins, PUMA and Noxa. In T98G cells, bortezomib-induced expression of caspase 4, documented after 24 h of treatment, with further activation of caspase 3, observed after 48 h of treatment. The delay in activation of caspase 3 correlated well with the delay of death of T98G cells. Our results do not support the possibility about direct cytotoxicity of aggregates of ubiquitin-conjugated proteins. They are more consistent with a view that proteasome inhibition is associated with both transcription-dependent and -independent changes in expression of pro-apoptotic proteins and consequent cell death initiation associated with caspase 3 activation.